Former investigations from our laboratory revealed that skeletal cells synthesize insulin-like growth factor (IGF) I, and its synthesis is regulated by CCAAT/enhancer binding proteins (C/EBP). Consequently, we explored the regulation and function of C/EBPs in osteoblastic cells and found that C/EBPs determine the cellular fate of undifferentiated stromal cells. We are particularly interested in the function of C/EBP homologous protein (CHOP) or C/EBP Zeta since its constitutive overexpression decreases adipogenesis and induces osteoblast differentiation in stromal cell cultures. This effect involves sensitization of the bone morphogenetic protein/Smad signaling pathway. The objective of this project is to understand the function of CHOP in bone in vivo and in vitro. For this purpose, we obtained chop null mice and are creating transgenic mouse lines overexpressing CHOP under the control of the type I collagen and of the osteocalcin promoters. Our initial studies demonstrated that chop null mice exhibit decreased bone formation. Furthermore, CHOP is required for normal osteoblastic function in vivo and in vitro, and chop null cells exhbit impaired collagen synthesis. Our specific aims are to extend these initial observations: 1) to determine the function of CHOP in vivo by transgenic overexpression of the nuclear protein under the control of the type I collagen or of the osteocalcin promoter so that expression occurs in differentiated cells and in osteoblasts in the bone microenvironment. The skeletal phenotype of transgenic mice will be compared to that of wild type mice and determined by histomorphometry, contact radiography, densitometry and micro CT scanning; 2) to determine the function of CHOP in vivo by defining the skeletal phenotype of chop null mice. This will be assessed with histomorphometric and radiological techniques, and if indicated, by examining structural and crystal properties of the skeleton; and 3) to determine the mechanism of action of CHOP in vitro, by overexpressing CHOP in cells of the osteoblastic lineage and by examining the phenotype of chop null cells. The impact of CHOP on the differentiation, function and apoptosis of cells of the osteoblastic lineage will be determined, and mechanisms involved and specific domains of the chop gene responsible for an effect will be defined. These investigations should clarify the role of CHOP in bone cell differentiation and function. [unreadable] [unreadable]